A major limitation of current ex vivo gene therapy protocols using the liver is that under most circumstances, little if any proliferation of transduced hepatocytes occurs after their transplantation, severely limiting levels of foreign gene expression. Recently, we developed a new model for liver repopulation in the rat in which more than 99% of endogenous hepatocytes can be replaced with transplanted cells. Extensive liver repopulation can be achieved with as little as 10,000 transplanted cells, and we are using this model to study the lineage and proliferative potential of fetal liver cells, hepatocyte progenitor cells, and liver-derived epithelial cell lines. We have initiated studies using this model for ex vivo gene therapy by transducing primary cultures of hepatocytes, hepatocyte progenitor cells and fetal hepatoblasts with lentivirus containing a reporter gene, green fluorescent protein (GFP), and then transplanting the transduced cells into the liver of suitably prepared hosts. Our fist hypothesis is that hepatocyte progenitor cells or fetal hepatoblasts will have a higher efficiency of viral transduction than primary adult. Our second hypothesis is that progenitor cells or fetal hepatoblasts will be superior to hepatocytes in maintaining long-term expression of transduced genes after they are transplanted into the liver. Our third hypothesis is that amplification of lentivirus transduced cells in the host liver will result in a high level of foreign gene expression throughout the organ, and thus achieve a level of expression suitable for effective gene therapy. These hypotheses will be tested in our new rat hepatic cell transplantation model using lentivirus-GFP. Once optimal conditions for effective gene transfer and expression are achieved, we will use this approach in an attempt to correct defective copper metabolism in the Long Evans Cinnamon (LEC) rat, a model for Wilson's disease, using a cloned Wilson's disease gene. Ultimately, we hope to adapt both cell transplantation and ex vivo gene therapy methods to humans with various inherited and acquired liver diseases, as these approaches are complementary but will have different clinical applications.